The rapid detection and identification of medically important organisms and macromolecular entities such as bacteria, viruses, malignant cells and the like is of critical importance in establishing diagnoses, treating patients, tracing the sources of infections, detecting biological contamination, and routinely screening and monitoring blood, other tissues and food so that public health might not be compromised.
The ability to detect and identify pathogenic organisms and macromolecular entities is limited by the sensitivity and rapidity of the detection system. In addition, identification of pathogenic organisms in blood or tissue samples poses special problems; not only is the availability of sample limited but the concentration of the pathogenic organism in that sample is often very low.
Techniques based on molecular methods of detection such as nucleic acid hybridization, restriction enzyme analysis, Southern analysis, Northern analysis, Western analysis and immunoassay have not overcome the problem of detecting low levels of pathogenic entities in dilute conditions. In many cases it is necessary to first incubate samples suspected of containing a pathogenic organism so as to enrich and increase the number of organisms to identifiable levels before detection and identification are possible (Andrews, W. H., Food Tech. 39:77-82 (1985)). However, growth and enrichment steps are extremely time-consuming in situations where time is of the essence to establish the presence or identification of an infectious organism.
In addition, growth requirements for some organisms are very complex and false negatives are a concern. Lack of growth of a bacterium may only indicate that the growth conditions weren't favorable, or that other, nonpathogenic bacteria in the sample grew faster than the organism in question and successfully "competed it out".
Methods for the detection and identification of pathogenic entities such as viruses are even more complex than those for entities like bacteria. More commonly, they depend on the acute and convalescent measurement of a serologic or antibody response to the infectious agent. These measurements are often time-consuming. They often depend on the identification and use of a suitable cell line which the virus can infect and in which the virus can replicate. They may also depend on the identification of an animal host which the virus can infect and in which the virus will induce diagnostic, serological symptoms.
Thus the identification of a pathogenic organism in blood and tissue samples may be missed even though the organism is present in the sample at levels infectious to humans.
Specific affinity reagents such as high affinity monoclonal antibodies have, in some cases, made it possible to confirm the presence, in blood or tissue samples, of organisms known or suspected of being infectious or otherwise pathogenic. For example, high affinity monoclonal antibodies directed to the hepatitis B surface antigen (HB.sub.s Ag) have been developed (Wands, J. R., et al., Gastroenterology 80:225-232 (1981)). These antibodies have successfully identified low levels of hepatitis B virus or its variants in the blood and tissues of some patients with acute and chronic liver disease but without known serologic markers of recent or past hepatitis B infection and also in some "healthy" individuals without clinical symptoms (Ben-Porath, E. et al., Progress in Liver Diseases 8:347-366 (1986); Ben-Porath, E. et al., J. Clin. Invest. 76:1338-1347 (1985)).
However, studies using monoclonal antibodies have been limited because it has been impossible to further characterize or study the molecular identity of the virus or variant in these patients. Levels of the virus, although detectable with the monoclonal antibody, are often too low for cloning, sequencing and other methods of viral characterization (See, e.g., Dienstag, J. L. et al., in Harrison's Principles of Internal Medicine, R. G. Petersdoff et al., eds., tenth edition, 1983, pp.1789-1801, McGraw-Hill, New York, incorporated herein by reference).
Current methods of identifying hepatitis B virus or its variants have depended on in vitro culture of the virus, radioimmunoassay, or genomic type identification after extraction of the vital DNA or RNA. However, these techniques do not always provide the necessary sensitivity for medical screening, diagnostic or treatment purposes (Id.). In addition, methods like radioimmunoassay may non-specifically detect the presence of viral antigens without providing information about the specific subtype.
The polymerase chain reaction (PCR) is a powerful technique for the amplification of specific DNA sequences (Cohen, S. N., U.S. Pat. No. 4,293,652; Erlich, H. A. et al., EP 258,017; Mullis, K. B., EP 201,184; Mullis et al., EP 200,362; Saiki, R. K., et al., Science 239:487-491 (1988); Mullis, K. B. et al., Meth. Enzymol. 155:335-350 (1987); Scharf, R. K., et al., Science 233:1076-1079 (1986) and Saiki, R. K., et al., Science 230:1350-1354 (1985)).
PCR has the ability to amplify a DNA sequence several orders of magnitude in a few hours, and has been used for: the detection of low levels of viral sequences (Kwok, S. et al., J. Virol. 61:1690-1694 (1987)), including hepatitis B (Kaneko, S., et al., Hepatology 8:1222 (1988)); cloning of low-abundant DNA sequences (Lee, M.S., et al., Science 237:175-178 (1987) ); the detection of malignant cells with chromosomal rearrangements (Lee, M.S., et al., Science 237:175-178 (1987)); the amplification of somatic mutational activation of cellular oncogenes in human tumors (Almoguera, C., et al., Cell 53:549-554 (1988)); and the detection and identification of individual DNA genotype in clinical and forensic samples (Marx, J. L., Science 240:1408-1410 (1988)), and haplotype (Li, H. et al., Nature 33.5:414-417 (1988)). The use of the PCR as a DNA diagnostic technique has been recently reviewed (Landegren, U., et al., Science 242:229-237 (1988), incorporated herein by reference).
PCR is based on the use of oligonucleotide primers, complementary to sequences flanking a particular region of interest, for primer-directed DNA synthesis in opposite and overlapping directions. With repeated cycles of high-temperature template denaturation, oligonucleotide primer reannealing, and polymerase-mediated extension, DNA sequences can be faithfully amplified several hundred-thousand fold. The amplified sequences are-remarkably accurate so one can reliably determine the nucleotide sequences immediately after PCR.
In theory, only one copy of the target gene need be present in a sample for PCR to adequately target and amplify it. For example, PCR amplification technique has been used to analyze the DNA in an individual diploid cell and a single sperm (Li, H., et al., Science 335: 414-417 (1988)). Ou, C. Y., et al., have suggested the use of PCR for the detection of HIV-1 virus in DNA from peripheral blood mononuclear cells (Science 239:295-297 (1988)).
However, use of PCR is not immediately applicable to all samples. For example, it is not possible to directly test blood or serum using PCR because serum contains many inhibitors of this technique. Studies utilizing PCR to investigate blood cells have had to first isolate DNA from the cells by phenol or other similar, suitable techniques known in the art for isolation and concentration of DNA. This results in a large loss of sensitivity.
Thus, there remains a need for methodology, applicable to serum and other biological samples, for the rapid identification of low levels of pathological entities. Such methodology would not require DNA isolation or prolonged incubation in vitro, would be sensitive enough to detect the presence of extremely dilute levels of organisms and macromolecular entities in a sample and would permit the cloning and genetic analysis of the pathological entity. Such methodology would still be technically simple enough to be embodied as a kit, and amenable for use as a routine screening method.